Chronic fungal infections are often recalcitrant to treatment. The recalcitrance of chronic infections is not well understood, but likely involves multiple factors including biofilm formation, and slow growth. In addition, diverse studies in different systems from bacterial infections to human tumors, found that populations can have heterogeneous drug susceptibilities even before treatment is initiated, leading to treatment failures. Heterogeneity within populations of fungal pathogens has not been well studied. We have identified chronic high burden C. lusitaniae lung infections in three unrelated subjects with cystic fibrosis (CF). C. lusitaniae has been documented in CF lung infections previously, but infections dominated by C. lusitaniae are not widely reported. In all three subjects, C. lusitaniae isolates within each population showed high variance in antifungal sensitivity, measured as the minimum inhibitory concentration (MIC), with some isolates stably resistant. We characterized the isolates from one subject further and discovered, through whole genome sequencing, that variable MICs correlated with heterogeneity in alleles of MRR1, a gene associated with clinical drug resistance in other Candida spp. exposed to drugs. Variation in MRR1 alleles within a single population was surprising as this subject had not been prescribed antifungals in the preceding year. Because of the ease of manipulation of C. lusitaniae, a haploid, genetically tractable, mating competent, yeast, we propose to leverage these isolate collections to study the basis for drug resistance, factors that promote selection for increased drug resistance, and factors that promote heterogeneous population structures in the lung. In Aim 1, we will test the hypothesis that substitutions in the Mrr1 central regulatory domain and Mrr1 C-terminal truncations lead to increased activity (1.1), that common and distinct genes are controlled by different classes of Mrr1 variants (1.2), and that differentially expressed Mrr1-regulated genes contribute to fluconazole resistance (1.3). In Aim 2, we will test the hypothesis that specific SNPs impact metabolic diversity (2.1), high Mrr1 activity promotes fitness in isolates with high rates of glycolysis (2.2), and that metabolic diversification promotes heterogeneity in drug resistance in nutritionally complex environments (2.3). Because of the propensity of C. lusitaniae to develop resistance to potent drugs like amphotericin B, often the antifungal of last resort, and the close relationship between C. lusitaniae and a recent multi-drug resistant fungal pathogen of concern, Candida auris, these studies are highly relevant to discovering ways to better understand the composition of chronic infections, to limit the development of antifungal resistance, and to combat resistant isolates once they develop.